Structure and Properties of a Bacterial Polysaccharide from a

Biomacromolecules 2003, 4, 1372-1379

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Structure and Properties of a Bacterial Polysaccharide from a Klebsiella Strain (ATCC 12657) O. Guetta, M. Milas, and M. Rinaudo* Centre de Recherche sur les Macromole´ cules Ve´ ge´ tales, CNRS, associated with Joseph Fourier University, BP 53, 38041 Grenoble Cedex 9, France Received April 28, 2003; Revised Manuscript Received June 24, 2003

The chemical structure and the rheological behavior of the Klebsiella polysaccharide ATCC 12657 was studied and compared with data described in the literature and obtained for similar polysaccharides. The acetylated polysaccharide presents in solution a normal viscoelastic behavior with no evidence of an ordered conformation whatever the experimental conditions are. The deacetylated form can induce the formation of physical gels, in the presence of salt excess or ethanol. Microcalorimetry, optical rotation, and rheology experiments demonstrate that a thermally reversible and highly cooperative conformational transition occurs at the same temperature than a sol-gel transition. The melting of the gel and the conformational transition temperatures are dependent on the nature of cations and ionic concentration, whereas the gel strength is only influenced by polymer concentration. Introduction The polysaccharide investigated in this work is produced by a Klebsiella aerogenes strain (referred as ATCC N°12657) formerly named Aerobacter aerogenes strain A3;1 a first chemical structure for the excreted polysaccharide, named K 54, was established at that time. The structure of the repeating unit was reexamined later2,3 and proposed as

It consists of a tetrasaccharide containing one L-fucopyranose, two D-glucopyranoses, and one D-(glucopyranose)uronic acid. From Dutton et al.2 and Franzen et al.3 the polysaccharide has acetyl group at O-2 of alternate L-fucosyl residues and a formyl substitution at O-4 of each lateral D-glucosyl group. Later, Dell et al.4 claimed that formyl was not included in the repeating unit. This polysaccharide is then a charged polymer and behaves as a polyelectrolyte in aqueous solution. From computer modeling, Elloway et al. described this polysaccharide as a right-handed 3-fold helix5 but on the basis of the structure initially proposed.1 Results obtained on a polysaccharide named XM-6, produced from an Enterobacter species, show that its chemical structure is the same as that of K54 but without any substituent.6 Atkins et al.7 have compared the X-ray * To whom correspondence should be addressed.

diffraction of XM-6 with that of K54 native or deacetylated. They concluded that the helical structure looks the same for two polysaccharides when they are non acetylated; XM-6 forms a double helix in which each individual chain has 8 sugar units per 3 turns of the helix. In the case of K54, the O-acetyl decoration inhibits association between molecules and prevents gelation; Atkins et al. mention that deacetylation causes a transition from 3-fold to 8-fold helix. Nevertheless, the X-ray pattern of K54 indicated a poorer cristallinity than that of XM-6, and the conclusion is not clear on the K54 conformation.7 On the opposite, XM-6, which is free of acetyl, forms gels in the presence of salt excess over a polymer concentration of 0.05% in the presence of Ca2+ ions and 0.3% in monovalent electrolytes. The sol-gel transition in this case is associated with a conformational coil-helix transition, which is reversible and very cooperative.6 In this paper, the characteristic behavior of the K54 in the native form, when it behaves as a thickening polymer, and after deacetylation, when it forms a physical gel, will be investigated. Experimental Section The polysaccharide was recovered from the fermentation broth, given by Solabia Cy. (France), after an enzymatic treatment with alcalase to hydrolyze the proteins. This treatment induces a clarification of the solution but causes a slight decrease of acetyl content. Then, the polysaccharide was precipitated with ethanol in the presence of NaCl to obtain the sodium salt form of the polymer. This polymer is considered as the purified native polysaccharide examined in the following. The deacetylated polysaccharide was obtained from the alcalase treated polysaccharide, by alkaline treatment in 0.1 M NaOH at 70 °C for 1 h. The solution was then neutralized,

10.1021/bm030036u CCC: $25.00 © 2003 American Chemical Society Published on Web 08/06/2003

Bacterial Polysaccharidefrom a Klebsiella Strain

Biomacromolecules, Vol. 4, No. 5, 2003 1373

Figure 1. 1H NMR spectrum for the initial polysaccharide (in absence of alcalase treatment) at 358 K, 300 MHz, in D2O and Ø 5 mm NMR tube.

filtrated, and concentrated and the polysaccharide recovered after freeze-drying. NMR experiments demonstrate that deacetylation is complete in the adopted conditions. The structure of the polysaccharide was tested by NMR in D2O at 80 °C using a Bruker AC300 spectrometer. Chemical shifts are given relative to external tetramethylsilane (TMS ) 0 ppm) and calibration was performed using the signal of the residual protons of the solvent as a secondary reference. Partial hydrolysis (pH ) 2.6 at 90 °C for 2 h) were performed to get a better resolution. Composition of the polysaccharide was determined by complete hydrolysis, by heating in acidic conditions (TFA 2 N, 1 h at 100 °C). Quantitative determination of sugars (derivatized as their alditol acetate) is achieved by gas chromatography (Hewlett-Packard 5890A) fitted with a flame-ionization detector with a SP2380 column (30m × 0.53 mm i.d.) using He as carrier gas. The polymer is usually solubilized in water and added with the same volume of salt solution two times more concentrated than the final chosen concentration. Because the gels are thermoreversible, and to avoid the gel formation during addition of salt with the deacetylated polymer, the system was heated to homogenize and then cooled to 4 °C before experiments. Different equipments were used to study the rheological behavior of gels and solutions: an Ubbelohde tube (with a capillary diameter of 0.58 mm), a coaxial Low Shear 40 (from Contraves) covering shear rate range from 0.01 to 200 s-1, a cone-plate AR1000 (from TA Instrument) covering shear rate range from 0.01 to 400 s-1, and frequency in dynamic experiments from 0.05 Hz up to 10 Hz. The coneplate rheometer is used with a cone of 4 cm diameter with 3°59 cone angle and a solvent trap to prevent solvent evaporation during heating the solution on the plane. The linear regime (G′ and G′′ independent of the strain amplitude imposed) is determined, before measurement, for dynamic experiments. The conformational enthalpy change is determined with a DSC III from SETARAM equipped with two 0.9 mL cells, one being filled with the solvent and the other with the

solution to test; the temperature variation is imposed at 0.4 °C/min. The rotary power determination was obtained on a PerkinElmer polarimeter 341 associated with a thermoregulator F3 Haake and a PG20; the quartz cell has a 10 cm path length, and the temperature variation was 0.4 °C/min. The wavelength was fixed at 360 nm. Results and Discussion A. Chemical Structure. Analytical and NMR experiments were performed to confirm that the structure of the polysaccharide ATCC 12657 is the same that the one described in the literature for K54. Gas chromatography experiments, realized on the monomeric units obtained after complete hydrolysis, and methylation indicate that the repeating unit of the polysaccharide ATCC 12657 is constituted of one fucopyranose, two glucopyranoses, and one (glucopyranose)uronic acid in agreement with the structure of K54. The nature and the configuration of the different sugars of the polysaccharide were controlled by 1H and 13C NMR spectroscopy. In Figure 1, the 1H NMR spectrum, obtained on the initial polysaccharide (absence of alcalase treatment), is shown; this sample was chosen to avoid any partial hydrolysis of the substituents. Signals around 2 and 1 ppm are characteristic of the 3 protons of the methyl group of the acetyl group and the fucose unit, respectively. The ratio of the two integrals gives 0.6 acetyl per repeating unit. No signal relates the presence of formyl group (at around 8 ppm in the 1H spectrum and 160 ppm in the 13C spectrum). The 1 H and the 13C spectra obtained on the partially hydrolyzed deacetylated polysaccharide are given in Figures 2 and 3. The presence of characteristic signals (-CH3 of fucose in the 1H spectrum at 1.05 ppm, C6 of the uronic acid at 176 ppm and the C-6 of two glucoses at 63 and 61 ppm in the 13 C spectrum) confirms the nature and ratio of the different sugars. The values of the coupling constants J1,2 of 4 Hz for the R-D-glucuronic acid and R-L-fucose and 7 Hz for the two β-D-glucose allow the determination of the configuration

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Figure 2.

1H

Figure 3.

13C

Guetta et al.

NMR spectrum for the deacetylated polysaccharide partially hydrolyzed, in D2O at 358 K and 300 MHz in a Ø 5 mm NMR tube.

NMR spectrum at 75 MHz in D2O at 358 K and 300 MHz in a Ø 5 mm NMR tube.

of the different sugars. The chemical shift of the two glucose C-6 signals confirms that the in-chain D-glucosyl residue is not linked through O-6 as first proposed by Sandford and Conrad.1 These results and the comparison with the data given in the literature for K54 confirm the structure of the polysaccharide given in the Introduction and the absence of formyl substituent. B. Physical Properties. The behavior of the two polysaccharides prepared (native and deacetylated) will be studied in the following. The data obtained on the deacetylated polysaccharide were compared with those given in the literature for XM6.

B-1. Acetylated Polysaccharide. A preliminary experiment by microcalorimetry demonstrates that no conformational change can be observed independently of the temperature and of the ionic concentration. Then, the conformation is assumed to be a coil in solution; this is confirmed in view of the well-defined NMR spectrum. Rheological experiments show a normal viscoelastic behavior observed for entangled polymeric solution.8 Because of the difficulties to prepare a dilute solution of the polysaccharide free of aggregates, it was impossible to get confident molecular weight determination from SEC experiments. The intrinsic viscosity [η] in 0.1 M NaCl at 25 °C is found equal 1550 mL/g and the



Figure 4. Relative viscosity in the Newtonian plateau of a solution of native polymer at 5 g/L, as a function of the external salt concentration (NaCl), at 5 and 25 °C.

Figure 5. Evolution of the relative viscosity of the native polysaccharide (concentration 5 g/L) in 0.4 M NaCl as a function of temperature on heating ([) and cooling (]). Temperature variation 0.4 °C/min, shear rate for measurements ) 0.02 s-1.

Huggins constant is k′ ) 0.66. The high value of k′ seems to indicate the presence of associations in solution. The molecular weight must be in the range of 1 × 106 by comparison to the characteristic of similar polysaccharides.9 The viscosity was studied as a function of NaCl concentration at two temperatures (5 and 25 °C), at 5 g/L for the native polysaccharide (Figure 4); it is clear that the large increase of the viscosity is induced in the semidilute solution by salt addition (over 0.1 M) and especially at low temperature. This indicates that molecular associations exist at low temperature in the presence of external salt screening the electrostatic repulsions. A continuous evolution of the viscosity at low shear rate (0.02 s-1) was investigated in the range of temperature from 5 to 40 °C at 0.4 °C/min (Figure 5). A hysteresis is demonstrated (at temperatures lower than 32 °C), but no change in the conformation in this range of temperature was observed by microcalorimetry (may be due to a lack of cooperativity and/or of sensitivity). These data indicate that heterogeneous multichain aggregates are formed at lower temperature. From these results, it comes that the acetylated polysaccharide is a coil in salt solution but that loose interchain

Figure 6. Melting temperature and helix-coil transition temperature determined on heating on a 3 g/L solution in 0.25 M NaCl by (a) Rheology (G′ at 5% strain and ω ) 0.1 Hz) (b) optical rotation at 360 nm (c) DSC.

interactions exist especially at a lower temperature and high salt content (larger than 0.1 M NaCl). This conclusion indicates that the helical conformation, existing in the solid state, is never stabilized in solution in the experimental conditions adopted in this work. B-2. Deacetylated Polysaccharide. The deacetylated polysaccharide, in mild conditions, becomes a gelling polymer in defined thermodynamic conditions. In Figure 6, the results obtained by rheology (G′ ) elastic modulus at 0.1 Hz), optical rotation, and DSC, as a function of temperature (on heating), allow us to evidence a conformational transition associated with a sol-gel transition around 30 °C for 3 g/L

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Ct. The results are given in Figure 8 and determine the phase diagram as usually for stereoregular biopolymers.12,13 The transition temperatures are highly influenced by the nature of cations and the ionic concentrations. A ionic selectivity is also observed. Conformations are more stable in the presence of NaCl or KCl than in the presence of TMACl. This result is similar to that obtained for XM66 and was attributed to the ionic radius of the cations. In Table 1, the enthalpies of conformational change (∆H) for the different experiments performed are given; the values are nearly constant and equal to ∆H # 4 kJ/mol. This value can be compared with the value calculated from the Manning14 and Record15 treatments relating the slopes of Figure 8 and ∆H: Figure 7. Evolution of the relative 1H NMR signal of fucose as a function of the temperature on ] cooling and [ heating in 0.4 M NaCl and evolution of the elastic modulus G′ on heating. Cp ) 5 g/L.

solution in 0.25 M NaCl. These transitions occur at the same temperature and are very cooperative, without hysteresis whatever are the experimental conditions. These conclusions are similar to what was found with the polysaccharide XM6;6 the gel can be related to the stabilization of a double helical structure in solution. The enthalpy of conformational change measured equals 4 kJ/mol (expressed per repeating unit); the experimental data are discussed later. In a separate experiment by 1H NMR, the evolution of the spectrum as a function of increasing temperature was studied in the presence of an internal standard (sodium succinate). The relative signal integral of fucose -CH3 is plotted in the same time as G′ in Figure 7. No signal exists for temperature lower than 33 °C, which is generally observed for ordered structure.10 Then it increases progressively because of the increase of the mobility of the chain over the conformational change. Remarkably, the NMR evolution is not as cooperative as that observed by rheology; this is not the same behavior as obtained with succinoglycan for which a cooperative transition is also observed by NMR.11 The behavior observed in this work may be caused by the double helix interactions causing the gel formation. Conformational Transition. The transition was examined in the range of polymer concentration from 0.5 up to 7 g/L and salt concentration from 0 to 0.5 M in NaCl, KCl, and TMACl. This will allow us to test the ionic selectivity and the role of the ionic concentration (Ct) on the temperature and enthalpy of conformational change. The total ionic concentration takes into account the amount of external salt added (Cs) but also the ionic concentration of the polymer (Cp). Considering the polysaccharide structure, the linear charge density equals 0.46, and then, Ct ) Cs + Cp. In the absence of external salt, no conformational change was observed for a polymer concentration lower than 30 g/L, i.e., Ct ) 0.045 equiv/L. This value corresponds to the screening of electrostatic repulsions allowing the stabilization of the helical conformation which is nevertheless less cooperative in the absence of external salt. The same type of results was previously observed for succinoglycan.11 The temperature for conformational change Tm (taken for half transition from DSC or optical rotation experiments) was investigated as a function of the total ionic concentration

∆H ) -2.3R/2(λh - λc) d(log CT)/d(1/Tm) where λh and λc ()0.46) are the charge parameters of the polysaccharide under the helical and coiled conformation. The ∆H values were calculated for a single (λh ) 0.51) or a double helical (λ2h ) 0.92) conformation assuming an extended chain conformation and based on the local conformation of polysaccharides; the data are compared in Table 2. The difference between experimental and calculated values does not allow us to conclude on the ordered conformation of the deacetylated K54 in solution; in fact, this model includes only the electrostatic contributions, but it is necessary to include nonpolyelectrolyte contribution, independent of the ionic concentration which modify the slope of the curves in Figure 8. It must be mentioned that because of loose interactions between the helical chains even, in absence of gel, it was not possible to determine the molar mass on both sides of the transition to demonstrate the existence of a single or double helix. Considering the literature data, it is considered a double helix; experimental ∆H values are lower than the calculated one (around 14 kJ/mol), and it is usually the case with other water soluble polysaccharides such as carrageenans or gellan that we have previously studied.6,16-18 Sol-Gel Transition. Figure 9 shows the behavior of the deacetylated polysaccharide in water and salt solutions at 5 g/L at 5 °C. In water, where there is no evidence of conformational transition, the evolution of G′ and G′′ is characteristic of viscoelastic solutions. In the presence of salt, at a temperature lower than the transition temperature, the behavior becomes characteristic of a gel (G′ > G′′ and nearly independent of the frequency). Increasing the concentration of polymer increases the gel strength (Figure 10) with a slope around 2, that is generally obtained for physical gels of polysaccharides.19 The gel strength (reflected by G′) seems to be only influenced by the concentration of polymer and not by the nature or the salt concentration even in the presence of divalent counterions (Figure 11). The role of salt concentration for two different polymer concentrations (1 and 5 g/L) is given in Figure 11; it shows that the G′ modulus is nearly independent of the salt concentration over 0.1 equiv/L of electrolyte. The gel appears earlier with divalent electrolyte (CaCl2), but there is no significant difference of the modulus between NaCl and KCl. The gel moduli are in agreement with the results given in Figure 12, showing that the reduced optical rotation, reflecting the



Figure 8. Evolution of the logarithm of the total ionic concentration as a function of the inverse of the temperature of conformational change (Tm-1). Tm is determined on heating by DSC in excess of different electrolytes. 2, TMA Cl; [, NaCl; b, KCl. Table 1. Transition Temperature and Enthalpies of Conformational Change Obtained by Microcalorimetry on Heating in Different Experimental Conditions

salt TMACl

NaCl

KCl

salt conc. (mol/L)

polymer conc. (g/L)

total ionic conc. (equiv/L) (×10-3)

0.05 0.1 0.2 0.4 0.05 0.1 0.25 0.4 0.07 0.1 0.25 0.4 0.05 0.1 0.25 0.4 0.05 0.1 0.25 0.4

3.1 3.1 3.1 3.1 1 1 1.1 1 5 5 5.1 5 1 1 1 1 5 5 5.1 5

54.7 104.7 204.6 404.6 51.5 101.5 251.7 401.5 77.4 107.5 207.6 407.5 51.5 11.5 251.5 401.5 57.5 107.5 257.6 407.5

Tm (°C)

∆H (kJ/mol)

7.5 12.5 19 27 10 18.8 29.5 34.8

n.d. 3.7 5 4.5 3.1 3.75 4.4 4.7 3.65 4.5 n.d. 4.7 n.d. 3.9 n.d. n.d. 3.5 3.9 4.4 4.5

19.3 29.8 35 10.6 19 29.2 34.1 12 19.8 29.7 34.5

degree of conformational order, is independent of the total ionic concentration (Ct is calculated for different polymer and electrolyte concentrations). An important characteristic for the gels formed is the melting temperature of the gels as shown in Table 3. As said previously, they correspond to the conformational transition temperature obtained in the same experimental conditions and depend only on the nature and salt concentration.

Table 2. Calculated Values for the Enthalpy of Conformational Change Considering a Double and a Single Chain Helical Structurea

salt

slope

∆H (kJ/mol) (calculated for a double helix)

NaCl KCl TMACl

-3136 -3300 -3480

13.8 14.5 15.3

a

∆H (kJ/mol) (calculated for a single helix) 2.1 2.2 2.3

The slopes of phase diagram from Figure 8 are also given.

Figure 9. Rheological behavior of polysaccharide (G′ full signs; G′′ empty signs) as a function of the frequency at 5 °C for 10% strain. Polymer concentration 5 g/L in water ([, ]) and in 0.25 M NaCl (b, O).

At polymer concentrations lower than 0.5 g/L, the gel behavior was not observed. However, Figure 13 shows that associations exist in solution causing large increase of the viscosity with a transition in the range of 27 °C corresponding to the induction of the conformational change and interchain interactions.

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Figure 10. Elastic modulus (G′ at 0.5 Hz) in 0.1 equiv/L salt excess at different polymer concentrations at 5 °C. Slope ) 2 ( 0.1.

Figure 12. Evolution of ∆R/Cp (∆R is the optical rotation difference between helix and coil conformations; Cp is the polymer concentration in g/L) as a function of the salt concentration in NaCl, [; KCl, b; and CaCl2, 9. Table 3. Relation between the Temperature of Gel-Sol Transition and the Nature of the Salt at a Constant Ionic Concentration (Cs ) 0.1 equiv/L)a salt

slope (n)

melting temperature (°C)

TMACl NaCl KCl MgCl2 CaCl2

2.08 1.89 1.81 1.71 2.04

12 18 18 27 29

a The slope (n) relating G′ and the polymer concentration in log-log plot (see Figure 10).

Figure 11. Evolution of the elastic modulus (at 0.5 Hz and 10% strain) as a function of the salt concentration for two polymer concentrations (1 and 5 g/L) at 5 °C. 2, CaCl2; 9, NaCl and KCl.

At end, it was demonstrated that stable gels are formed in the presence of ethanol up to 50% (volume fraction). The polysaccharide was dissolved in water, and ethanol was progressively added. When ethanol is added to 20% (V/V) in absence of external salt, a gel is formed; the conformational transition temperature of these gels obtained by DSC are given in Table 4; the helical conformation is more stable than that obtained in pure aqueous solution, at the same polymer concentration. Over 50% (V/V ethanol/water), the polysaccharide phase separated. The addition of EtOH decreases the solubility parameter of the polymer and favors the interchain interaction promoting the gelation. This phenomena is promoted by salt addition, screening the eletrostatic repulsions as mentioned in Table 4. Rheological properties of the gels obtained at 3 g/L polymer in 20% ethanol in the presence of different salt concentrations give G′ ) 17 Pa independently of salt and ethanol concentrations. This value is nearly equal to that obtained in aqueous solution at the same polymer concentration. Then, it is concluded that polymer concentration is the most important parameter controlling the gel mechanical properties. These results are interesting in view of new applications for this original polysaccharide.

Figure 13. Relative viscosity (at 10 s-1 shear rate) as a function of temperature for a dilute solution (0.3 g/L) in 0.25 M NaCl.

Conclusion This paper allows us to compare the behavior in solution of the native K54 type polysaccharide and that of the deacetylated form to demonstrate the importance of substituents on the physical properties of bacterial polysaccharides. It is clear that the acetyl substituents grafted along the chain prevent gelation but also destabilized the possible helix in acetylated K54 dissolved in aqueous solutions. Same role of the acetyl substituents, preventing the packing of double


Bacterial Polysaccharidefrom a Klebsiella Strain Table 4. Influence of Ethanol and NaCl Concentration on the Temperature of Helix-Coil Transition Obtained by DSCa

a

NaCl conc. (equiv/L)

EtOH % (vol.)

Tm (°C)

0 0 0 0.05 0.1 0.2 0.05 0.2

20 30 40 20 20 20 10 10

17 29 40 34 42 49 24 38

The polymer concentration is 3 g/L.

helices and gelation, was previously described for gellan.8 The structure of the Klebsiella polysaccharide ATCC 12657 was established and found identical to that of K54. In solution, it is a coiled chain, showing loose interchain interactions because of the acetyl groups and depending strongly on the temperature. After deacetylation, this polysaccharide becomes a gelling polymer in the presence of external salts; the conformational transition and the melting temperatures are similar and depend only on the nature and salt concentration, whereas the modulus of the gels is mainly depending on the polymer concentrations. Monovalent and divalent counterions give nearly the same modulus at a given polymer concentration. In addition, a gel is also formed in the presence of ethanol from 20 up to 50% ethanol (v/v ethanol/water) in the absence of external salt allowing to extend application of such a polymer. Acknowledgment. The authors thank Solabia Cy (France) for their financial help. References and Notes (1) Sandford, P. A.; Conrad, H. E. The structure of the Aerobacter aerogenes A3 (Sl) polysaccharide. I. A reexamination using improved procedures for methylation analysis. Biochemistry 1966, 5, 15081517. (2) Dutton, G. G. S.; Merrifield, E. H. The Capsular Polysaccharide from Klebsiella Serotype K54; Location of the O-acyl Groups, and a revised Structure. Carbohydr. Res. 1982, 105, 189-203. (3) Franze´n, L.-E.; Aman, P.; Darvill, A. G.; McNeil, M.; Alberstheim, P. The Structure of the acidic polysaccharide secreted by Klebsiella Aerogenes Type 54 Strain A3. Carbohydr. Res. 1982, 108, 129138.

(4) Dell, A.; Dutton, G. G. S.; Jansson, P.-E.; Lindberg, B.; Lindquist, U.; Sutherland, I. W. Absence of O-formyl groups in Klebsiella Polysaccharide. Carbohydr. Res. 1983, 122, 340-343. (5) Elloway, H. F.; Isaac, D. H.; Atkins, E. D. T. Review of the structures of klebsiella Polysaccharides by X-ray diffraction. In Fiber Diffraction; Frtench, A. D., Gardner, K. C., Eds.; ACS Symposium Series 141; American Chemical Society: Washington, DC, 1979; pp 429458. (6) O’Neill, M. A.; Morris, V. J.; Selvendran, R. R.; Sutherland, I. W.; Taylor, I. T. Structure of the Extracellular Gelling Polysaccharide produced by Enterobacter (NCIB 11870) Species. Carbohydr. Res. 1986, 148, 63-69. (7) Nisbet, B. A.; Sutherland, I. W.; Bradshaw, I. J.; Kerr, M.; Morris, E. R.; Shepperson, W. A. XM-6: A New Gel-forming Bacterial Polysaccharide. Carbohydr. Polym. 1984, 4, 377-394. (8) Atkins, E. D. T.; Atwool, P. T.; Miles, M. J.; Morris, V. J.; O′Neil, M. A.; Sutherland, I. W. Effect of acetylation on the molecular interactions and gelling properties of a bacterial polysaccharide. Int. J. Biol. Macromol. 1987, 9, 115-117. (9) Mazen, F.; Milas, M.; Rinaudo, M. Conformational transition of native and modified gellan. Int. J. Biol. Macromol. 1999, 26, 109118. (10) Guetta, O.; Mazeau, K.; Auzely, R.; Milas, M.; Rinaudo, M. Structure and properties of a new bacterial polysaccharide named Fucogel. Biomacromolecules 2003, 4, 1362. (11) Bryce, T. A.; McKinnon, A. A.; Morris, E. R.; Rees, D. A.; Thom, D. Chain conformations in the sol-gel transitions for polysaccharide systems, and their characterization by spectroscopic. Faraday Discuss. Chem. Soc. 1974, 57, 221-229. (12) Gravanis, G.; Milas, M.; Rinaudo, M.; Clarke-Sturman, A. J. Conformational transition and polyelectrolyte behaviour of a succinoglycan polysaccharide. Int. J. Biol. Macromol. 1990, 12, 195200. (13) Milas, M.; Rinaudo, M. Conformational investigation on the bacterial polysaccharide xanthan. Carbohydr. Res. 1979, 76, 189-196. (14) Burova, T. V.; Golubeva, I. A.; Grinberg, N. V.; Mashkevich, A. V.; Grinberg, V. Y.; Usov, A. I.; Navarini, L.; Cesaro, A. Calorimetric Study of the Order-Desorder Conformational Transition in Succinoglycan. Biopolymers 1996, 39, 517-529. (15) Manning, G. S. Limiting laws and counterions condensation in polyelectrolyte solutions. I. Colligative Properties. J. Chem. Phys. 1969, 51, 924-934. (16) Record, M. T. J. Effects of sodium and magnesium ions on the helixcoil transition of DNA. Biopolymers 1975, 14, 2137-2158. (17) Milas, M.; Shi, X.; Rinaudo, M. On the physicochemical properties of gellan gum. Biopolymers 1990, 30, 451-464. (18) Boutebba, A.; Milas, M.; Rinaudo, M. Order-Disorder. Conformational transition in succinoglycan: calorimetric measurements. Biopolymers 1997, 42, 811-819. (19) Rochas, C.; Rinaudo, M. Mechanism of gel formation in kappacarrageenan. Biopolymers 1984, 23, 735-745. (20) Rinaudo, M. Gelation of polysaccharides. J. Intell. Mater. Syst. Struct. 1993, 4, 210-215.

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Structure and Properties of a Bacterial Polysaccharide from a

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